Research On Apoptosis Induced By Modified Shengma Biejia Decoction In AML Cells Via Regulating MAPK Pathway

Research Purpose:Based on our previous clinical trials we have demonstrated that the modified Shengma Biejia decoction(SMBJD)can significantly increase the effective rate of chemotherapy in elderly patients with acute myeloid leukemia(AML)and improve their quality of life.This topic before the pre-experiment also confirmed that SMBJD can significantly inhibit the proliferation of AML cells,but its specific mechanism of action is not clear.In this experiment,the effects of SMBJD on the proliferation and apoptosis of AML cells HL-60,NB-4 and K562 in vitro were studied,and its molecular mechanism was discussed in order to provide more scientific basis for the application of TCM in AML therapy.Research Methods:Research methods in vitro1 SMBJD(2,4,6,8,10 mg/mL)were used to intervene in HL-60,NB-4 and K562 cells at different end concentrations respectively,and the proliferative activity of each group of cells was detected by MTT experiment at different time pionts(24,48,72 h).The inhibition rate of cell proliferation was calculated.2 Annexin V/PI dual-dyeing flow cytometry(flow cytometry,FCM)test was used to observe the effects of SMBJD on cell apoptosis rate in HL-60,NB-4 and K562 cells.3 Hoechst33258 staining was used to observe the effects of SMBJD on morphological changes of apoptosis in HL-60,NB-4 and K562 cells.4 The effect of SMBJD on mitochondrial membrane potential of HL-60,NB-4 and K562 cells were observed by mitochondrial membrane potential detection in JC-1 staining cells.5 The effect of SMBJD on cell cycle of HL-60,NB-4 and K562 cells was observed by FCM.6 The effects of SMBJD on HL-60 cells apoptosis and MAPK signaling pathway-related protein expression immune imprinting experiment(Western Blot,WB),taking time and concentration as changing factors respectively.7 P38 inhibitors SB203580,JNK inhibitors SP600125 and ERK inhibitors U0126 were used to block MAPK signaling pathway respectively,and then the expression changes of apoptosis-related proteins was detected by WB experiment.Research methods in vivo1 HL-60 cells were cultured in vitro and used to establish a model of nude mice with tumor.When turmor formation,all model mice were divided into control group and experimental group randomly.The mice in experimental group was administered with SMBJD every day to observe the anti-leukemia effect in vivo.2 During the per:iod of SMBJD intervention,the mice body weight in each group were weighed every other day,and the changes in body weight and survival status of mice after anterior medicine were observed and compared.3 TUNEL(TdT-mediated dUTP Nick-End Labeling)staining kit was used to obsenve the apoptotic cells in tumor xenografts induced by SMBJD.4 Immunohistochemical assay(IHC)was used to detecte the expression of apoptosis-related proteins in xenograft tumor.Research Results:Results in vitro1 Results of MTT assay showed that at the same time point(24 h),with the increasing SMBJD concentration(2,4,6,8,10 mg/mL),the proliferation inhibition rates of HL-60,NB-4 and K562 cells were gradually increased,and the difference was statistically significant compared with the control group(P<0.01).The same concentration of SMBJD on each group of cells for 24 h,48 h and 72 h,respectively.The inhibition rate of cell proliferation increased with the prolongation of drug action time.Therefore,SMBJD has a significant inhibitory effect on the proliferation of AML cells,and the effect shows a good concentration and time dependence2 Flow cytometry was used to detect the apoptosis rate of HL-60,NB-4 and K562 cells treated with different concentrations of SMBJD(4,6,8 mg/mL).The results showed that SMBJD significantly increased the number of Annexin V(?)cells in each group and induced apoptosis.The apoptosis rate increased with the increase of drug concentration,which showed obvious concentration dependence.3 HL-60,NB-4 and K562 cells were treated with SMBJD at different concentrations(4,6,8 mg/mL)for 24 h respectively.After staining with the Heochest33258,it w'as obvious that apoptosis morphological manifestations,such as gradual nuclear shrinkage and fragmentation,could be observed.Compared with the control group,with the increase of SMBJD concentration,the number of cells with apoptosis morphological changes gradually increased,showing a significant concentration-effect dependence.4 After treatment of different concentrations of SMBJD(4,6,8 mg/mL)for 24 h,HL-60,NB-4 and K562 cells were stained with a mitochondria staning kit(JC-1)in order to detect the mitochondrial membrane potential by flow cytometry.The results of this experiment showed that SMBJD could reduce the mitochondrial membrane potential level of cells in each group.And in the comparison with the control group,with the increase of SMBJD concentration,the trend of cell mitochondrial membrane potential decline was also significantly enhanced.5 The results of cell cycle assay showed that SMBJD could cause HL-60 and NB-4 cells to block in the S phase,K562 cells block in the G2/M phase.And when compared with the control group,with the increase of SMBJD concentration,the proportion of cells in the block cycle of each group of cells increased correspondingly,showing a significant concentration dependency relationship.6 HL-60 cells were treated with different concentrations of SMBJD(4,6,8 mg/mL),and detected by Western blot after 24 h.The results showed that the expression of cleaved PARP and cleaved caspase-3,key proteins involved in apoptosis,up-regulated with the increase of SMBJD concentration.The expressions of cleaved-caspase-8 and cleaved-caspase-9,key proteins involved in apoptosis pathways and pro-apoptotic protein Bax were also up-regulated with the increase of SMBJD concentration,while the expression of anti-apoptosis protein bcl-2 was down-regulated.Meanwhile,the expressions of MAPK pathway related protein p-ERK,p-JNK and p-p38 also up-regulated with the increasing concentration of SMBJD.7 A medium effective concentration of SMBJD(6 mg/mL)was used to treat HL-60 cells for 6 h,12 h,18 h and 24 h,respectively.Western blot results showed that the expression of cleaved-PARP,cleaved-caspase-3,cleaved-caspase-8,cleaved-caspase-9 and pro-apoptotic protein Bax were up-regulated with the prolongation of drug action time,while the expression of anti-apoptosis protein bcl-2 was down-regulated.At the same time,the expression of p-ERK,p-JNK and p-p38 also up-regulated with the increase of SMBJD action time.8 HL-60 cells were pretreated with 18uM p38 inhibitor SB203580,20uM JNK inhibitor SP600125 and 20uM ERK inhibitor U0126 for 4 h before the treatment of SMBJD(6 mg/mL)to block the MAPK pathway.Western blot results showed that the expressions of apoptosis-related proteins cleaved-caspase-3,cleaved-caspase-8 and cleaved-caspase-9 in the each inhibitor combined with SMBJD intervention group were decreased when p3 8,JNK and ERX signals were inhibited respectively,compared with the SNMBJD intervention group.Results in vivo1 After 15 days of herbal administration,there was no significant arrest in the growth of the SMBJD-treated xenografts,although there were some visual differences compared to that in untreated control.2 During the course of drug administration,the weight of mice in both groups was weighed and recorded every other day.The statistical results showed that there was no significant difference in body weight between the SMBJD-treated group and the control group.3 TUNEL staining kit was used for staining xenografts sections of mice.The results of microscopic imaging showed that no obvious apoptotic cells were found in the control group,while a large number of tunel-labeled apoptotic cells were found in the xenografts sections of mice treated with SMBJD.4 When compared with the control group,the results of IHC assay showed that the expressions of the apoptosis-related protein cleaved-caspase-3 and pro-apoptotic protein Bax of SMBJD group were increased,while the expressions of the anti-apoptotic protein bcl-2 of SMBJD were decreased.Research Conclusion:1 SMBJD can significantly and effectively inhibit the proliferation of HL-60,NB-4 and K562 cells in vitro,and the effect presents concentration-time correlation.2 SMBJD could induce apoptosis of HL-60,NB-4 and K562 cells in vitro,and the process may involve the initiation of endogenous and exogenous apoptosis pathways,and the MAPK signaling pathway may be involved in the apoptosis process as an upstream regulatory signal.3 SMBJD can have periodic block effects on HL-60,NB-4 and K562 cells in vitro respectively,however,this effect does not show periodic specificity.4 SMBJD had no significant effect on the body weight and other living condition indexes of nude mice with HL-60 cell tumor.5 SMBJD had a certain inhibitory effect on the growth of tumor in HL-60 tumor mice,however,there was no significant statistical difference compared with the control group.6 The mechanism by which SMBJD inhibits the proliferation of AML cells in vivo may invole apoptosis induction.